Particle analysis in an acoustic cytometer

ABSTRACT

The present invention is a method and apparatus for acoustically manipulating one or more particles. Acoustically manipulated particles may be separated by size. The particles may be flowed in a flow stream and acoustic radiation pressure, which may be radial, may be applied to the flow stream. This application of acoustic radiation pressure may separate the particles. In one embodiment, the particles may be separated by size, and as a further example, the larger particles may be transported to a central axis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of the filing ofU.S. Provisional Patent Application Ser. No. 61/008,422, entitled“Particle Analysis in an Acoustic Cytometer”, filed Dec. 19, 2007, andthe specification thereof is incorporated herein by reference.

STATEMENT OF GOVERNMENT RIGHTS

The invention was made with U.S. Government support. The Government mayhave certain rights in the invention through the Department of Energyunder contract number DE-AC52-06NA25396.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

Embodiments of the present invention relate to acoustic cytometry andmore specifically to particle analysis in an acoustic cytometer.

2. Background

It was realized early in the development of flow cytometry that theangular dependence of the light scattering intensity from a particlecontains a wealth of information related to a particle's intrinsic andextrinsic properties. For example, Mullaney, et al. experimentallydemonstrated the use of forward light scatter (0.5-2 degrees) for cellsize estimation. In the same time period, it was also realized that cellorientation relative to the incident light beam can introduce artifactsthat affect population resolution and instrument sensitivity forparticles that do not possess axial symmetry parallel to the flowdirection. Loken et al. showed that nonspherical particles in the formof fixed chicken erythrocytes yield a bimodal scattering distributionthat is related to rim-on or face-on illumination of the disk-cellstructure. Particle orientation dependent scattering effects due toasymmetries that are apparent in the complex geometrical shape of spermcells have also been demonstrated. Several researchers have suggestedpassive solutions to orienting particles by shaping the sample nozzle tointroduce asymmetries into the velocity flow field of the hydrodynamicsheath system. It has been shown that the most critical aspect inefficient X and Y sperm discrimination in flow cytometric sorting is theorientation of the sperm in the optical scattering plane. Recently,novel nozzle geometries have demonstrated proper orientation of up 60%of the sperm heads in the optical scattering plane at analysis ratesnear 2000 particles/s dramatically affecting the sorting efficiency.Other researchers have addressed particle asymmetries by collecting dataover a large array of scattering angles using scanning flow cytometry(SFC) at the cost of lower particle analysis rates (approximately 400particles/s). Systems have demonstrated continuous angular scatteringdata spanning 70 degrees but the data is taken relative to an arbitraryparticle orientation that results in large variations of the collectedangular spectra for asymmetric particles.

One of the earliest large-scale demonstrations of separating biologicalcells using acoustic forces was done by Doblhoff, et al. In that system,acoustic radiation pressure was used for selective retention of viablehybridoma cells for the purpose of removing nonviable cells and smallercellular debris from a 20 liter bioreactor. That system was based on amulti-planar transducer design and demonstrated viable cell retentionrates as high as 99.5% with varying results for cellular debrisrejection. That early system required high power input (in excess of 15W) thus necessitating a cooling unit for the drive transducers. Morerecently, Feke and coworkers developed a novel particle separationstrategy that relies on both acoustic radiation pressure and secondaryacoustic forces. A high-porosity polyester mesh (pore size two orders ofmagnitude greater than particle size) in an acoustic standing waveserved as a collection matrix whereby particles at nodal locations weretrapped within the mesh and secondary acoustic forces formed particleagglomerates and created an attractive force at the mesh surface. In asimilar demonstration of retention of hybridoma cells, retentionefficiencies of about 95% were achieved with negligible effects on cellviability. This system achieved high cell densities of approximately1.5×10⁸ cells/mL with only hundreds of milliwatts of input power.

SUMMARY OF THE INVENTION

One embodiment of the present invention comprises a method foracoustically manipulating one or more particles. This embodimentpreferably includes flowing a fluid containing particles therein,applying acoustic radiation pressure to the fluid and acousticallymanipulating the one or more particles, stopping the fluid, andreviewing at least one of the particles. In this embodiment of thepresent invention reviewing at least one of the particles can includeanalyzing at least one of the particles, and/or sorting at least one ofthe particles. The particles can also be flowed through a flowcytometer.

Another embodiment of the present invention comprises a method foracoustically manipulating one or more particles. This embodimentincludes flowing a fluid containing particles therein, applying acousticradiation pressure to the fluid and acoustically manipulating the one ormore particles, reversing the direction of flow, and reviewing at leastone of the particles that were reversed in the flow. In this embodiment,reviewing the particles can comprise analyzing at least one of theparticles and/or sorting at least one of the particles. This embodimentcan also include flowing the fluid through a flow cytometer.

Yet another embodiment of the present invention comprises a method foracoustically aligning and orienting particles in a flow stream of aparticle analyzer comprising flowing particles through the flow stream,subjecting the particles to an acoustic radiation pressure, acousticallyaligning the particles in the flow stream of the particle analyzer, andacoustically orienting the particles in the flow stream of the particleanalyzer. In this embodiment, the particle analyzer can be a flowcytometer. This method can also include a non-axisymmetric field whereinthe particles are asymmetric. Further, this embodiment optionallycomprises selecting a predetermined orientation of the particles, wherethe orientation is within an optical scattering plane. This embodimentcan comprise distinguishing between different types of particles basedupon particle asymmetries. This embodiment can further comprise yieldingrepeatable orientations of particle clusters or sorting particles. Thesorting of the particles can include pre-analysis in-line separating ofparticles based on size. In addition, this embodiment can align theparticles in a polar direction about a flow axis or align the particlesin a direction of flow. Still further, this embodiment can optionallycomprise fractionating the particles. In this embodiment, the particlecan be a red blood cell, a platelet or a sperm.

Another embodiment of the present invention comprises a method foranalyzing particles in a particle analyzer. This method preferablycomprises flowing particles in a flow stream, applying a radial acousticradiation pressure to the flow stream, acoustically orienting non-axialsymmetric particles in the flow stream, transporting the particlesthrough an interrogation point, and analyzing the particles. Thisembodiment can also include aligning the particles in a flow stream witheither an acoustic field or a hydrodynamic focus. This method alsopreferably comprises concentrating the particles in the flow stream.

Still another embodiment of the present invention comprises a method ofanalyzing one or more particles in a flow stream. This embodimentpreferably comprises labeling the particles with a lanthanide, flowingthe particles in the flow stream, applying acoustic radiation pressureto the flow stream to maintain particle focus regardless of flow rate,and analyzing the particles with the lanthanide. This embodiment canalso include stopping and/or reversing the flow stream.

A further embodiment of the present invention comprises a method forsorting particles by size. This method preferably comprises flowingparticles in the flow stream, applying radial acoustic radiationpressure to the flow stream, and acoustically sorting and separating theparticles by size. This method also preferably comprises transportinglarger particles of the separated particles into a flow cytometer andanalyzing the larger particles. The transporting step of this embodimentpreferably transports the larger dependent particles to a central axisof the flow stream. This embodiment also preferably comprises in-linesorting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention. In the drawings:

FIG. 1 is an embodiment of the present invention illustrating a linedrive capillary where particles are acoustically focused to the centralaxis of the capillary;

FIG. 2A is a density plot of acoustic force potential in the crosssection of a circular capillary driven by a line source according to oneembodiment of the present invention;

FIG. 2B illustrates an induced particle rotation to lie in a stableforce equilibrium plane according to one embodiment of the presentinvention;

FIG. 3 is an embodiment of the present invention illustrating a linedriven acoustic fractionator where large particles are transported tothe capillary axis while smaller particles remain unaffected by theacoustic field;

FIGS. 4A and 4B illustrate particles flowing through a capillary in arandom orientation when the acoustic field is off and then particlesaligned coincident with the capillary axis upon excitation of theacoustic field according to one embodiment of the present invention;

FIGS. 5A-5C illustrate selective fractionation of particles that areapproximately 1 μm and approximately 10 μm in a line drive capillaryaccording to one embodiment of the present invention;

FIG. 6 is an embodiment of the present invention illustrating a sampleinput into a flow cytometer where the sample is concentrated reducingits volume and thus reducing the analysis time in flow cytometryapplications.

DETAILED DESCRIPTION OF THE INVENTION

As used herein “a” means one or more.

As used herein “capillary” means a flow channel or chamber having ashape selected from rectangular, elliptical, oblate circular, round,octagonal, heptagonal, hexagonal, pentagonal, and triagonal.

In one embodiment of the present invention, acoustic radiation pressurepreferably focuses particles to the center of a capillary. Thisembodiment enables fundamental changes in the way single cells orparticles can be either analyzed or processed in a flow stream. Thissample delivery embodiment expands the analysis capabilities of flowcytometry by improved analysis and/or pre-analysis sample preparationand purification.

A non-limiting embodiment of an acoustic particle focusing device isillustrated in FIG. 1. This device preferably comprises acousticallydriven capillary 10 where an acoustic particle trap oriented parallel tothe flow direction comprises line source 12 and fluid 18 withparticles/cells 16. This embodiment enables removal of high-velocitysheath flow associated with hydrodynamic focusing and allows extendedparticle transit times within the optical interrogation region whilemaintaining a high particle analysis rate due to an inherent in-lineparticle concentration effect. Additionally acoustic focusing of theparticle stream provides the ability to stop and reverse the flowdirection without defocusing the particle stream while maintainingparticle registration. The increased particle transit times provide anopportunity for high-sensitivity optical measurements that use low-powerlight sources and less sensitive optical components. Control of flowdirection and rate allows for reanalysis of targets of high significancethereby minimizing uncertainties in the scattering data associated withsystem level fluctuations.

An additional property of an acoustically driven flow chamber is anon-axisymmetric force field that is created within the flow stream.Force asymmetries within the flow chamber orient nonspherical cells orparticles such that consistent scattering signatures, that are notpossible with standard hydrodynamic flow systems, are produced bypositioning asymmetric particles in a predetermined orientation withinthe optical scattering plane. In this embodiment, optical scatteringdata associated with specific particle orientation can, for example,distinguish between different types of bacteria based upon particleasymmetries and improve analysis and sorting of irregular cell typessuch as RBC's and sperm cells. The asymmetric force field also yieldsrepeatable orientations of particle clusters that are formed from thecoalescence of multiple microspheres or cells (e.g. ‘dumbbell’ shapesfrom agglutination of two particles). Discrimination of particleclusters can be made easier by pulse shape analysis and angularscattering interpretation due to the unique induced orientation of thecluster relative to the scattering plane (e.g. central axis of the‘dumbbell’ always parallel to the flow axis).

An acoustically line-driven capillary embodiment of the presentinvention brings new modes of particle and cell analysis to thedetection system of a flow cytometer, and is also employed in particleor cell separations for in-line sample preparation. A primary advantageof acoustic radiation pressure is that it can be used in fairly largechambers with high volume throughput. The acoustic field acts similarlyon most biological particles and is thus inherently nonspecific wherebymost biological particles are transported to the same spatial location.The magnitude of the field, however, is highly size dependant and makesacoustic radiation pressure an excellent candidate in applications thatrequire high throughput, pre-analysis in-line separating of particlesbased upon size e.g. sperm from vaginal cells in forensic analysis,virus from bacteria or intact cells from cellular debris. In thedescription above, a line-driven capillary with a cylindrical geometryis used as the acoustic focusing device, but general geometry (square,rectangular, elliptical, oblate circular, etc.) devices that employacoustic radiation pressure to position the particles can be used inapplications of particle separation, debris rejection, particlealignment, and sample purification.

Acoustic Radiation Pressure

The force on a particle resulting from acoustic radiation pressuredepends upon frequency of excitation, pressure amplitude within themedium, and the density/compressibility contrast between the particleand the host medium. Within an acoustic standing wave, it is atime-averaged drift force which transports the particles to a nodal oran anti-nodal position. The expression for the acoustic radiation forcepotential U on a spherical particle in an acoustic standing wave isgiven by:

$U = {\frac{4}{3}\pi\;{\alpha^{2}\left\lbrack {{\left( {\beta_{o}\frac{\left( p^{2} \right)}{2}} \right)f_{1}} - {\frac{3}{2}\left( \frac{\rho_{o}\left( v^{2} \right)}{2} \right)f_{2}}} \right\rbrack}}$

Here, α is the particle radius, β₀ is the compressibility of thesurrounding fluid, and ρ₀ is the density of the surrounding fluid. Thepressure and velocity of the acoustic field in the absence of theparticle are described by p and v, respectively, and the bracketscorrespond to a time-averaged quantity. The terms f₁ and f₂ are thecontrast terms that determine how the mechanical properties of theparticle differ from the background medium. They are given by:

$f_{1} = {1 - \frac{\beta_{p}}{\beta_{o}}}$$f_{2} = \frac{2\left( {\rho_{p} - \rho_{o}} \right)}{\left( {{2\rho_{p}} - \rho_{o}} \right)}$

The subscript ρ corresponds to intrinsic properties of the particle. Theforce F acting on a particle is related to the gradient of the forcepotential by:F=−∇U

Particles are preferably localized at positions where the potential Udisplays a minimum. (For a circular cross section capillary, a potentialminimum is coincident with the axis of the capillary forming theparticle trap in FIG. 1 when driven in a dipole type mode. Other modesexist and are advantageous for spatial positioning of particles inlocations other than the axis of the capillary for selectedapplications.)

Acoustic Line-Driven Capillary

Forces resulting from acoustic radiation pressure are preferably aneffective means to localize particles in an arrangement similar tohydrodynamic focusing without the need for sheath fluids. Theline-driven capillary of the present invention has been proven effectivein sheath replacement. (A capillary with a source aperture larger than aline contact can yield similar results. This embodiment has demonstratedacoustically driven capillaries with source apertures that have anextended contact length along the circumference of the capillary thatspans more than about 45 degrees.) It is constructed from a capillarythat is driven by a piezoceramic source in contact with its outer wall.Vibration of the structure creates a localized pressure node along thecentral axis where an axial particle trap is formed. A diagram of thisdevice is given in FIG. 1. Particles in a dilute suspension enter thedevice from the top and experience a radial force that transports themto the pressure node as they flow through the system. In an embodimentof the present invention, the particles contained in a sample aresimultaneously concentrated and aligned in single file as they are thentransported through the interrogation laser. The particles aretransported through the interrogation laser via various transportdevices, including but not limited to, a pump and/or one or more valves.

Implementation of acoustic particle focusing preferably allows new flowcytometry techniques and methods to evolve due to fundamental changes inthe way particles are positioned within the sample cell. Concentric flowstreams with different flow velocities are not required as withconventional hydrodynamically sheath-focused systems. Acousticallyfocused sample streams can be stopped, slowed, reversed or anycombination thereof without degrading alignment of the particle streamwithin the flow chamber. The increased residence time within theacoustic field produces a stream of particles whose focus is actuallyimproved. Additionally, the flow can be reversed with no adverse effecton particle alignment within the flow chamber allowing rare targets tobe repeat analyzed or stopped for extended analysis such as spectraldecomposition of the scattered/fluorescence signature.

One of the unique flow capabilities of the present invention is theability to select the sample delivery rate. By slowing cell/particletransit times (approximately 20-100 times slower than conventionalsystems) higher sensitivity optical measurements and measurements ofphotonic events that require longer interrogation times such asluminescence are possible.

Particle Orientation in a Standing Acoustic Wave Field

A known orientation of a particle as it passes through the interrogationregion enables light scatter/fluorescence measurements that providesignificant insight into cellular structure and intrinsic opticalproperties. The removal of several degrees of rotational freedom provesan invaluable tool to flow cytometry by increasing the value of currentlight scatter measurements by calibrating them to a specific orientationof a cell/particle and allowing reasonable consideration of new scatterangles as measured parameters. The acoustic line-driven capillary of thepresent invention (or other methods to introduce acoustic radiationpressure into the flow cell) is an active means to rotate and alignparticles in both the direction of flow and in polar directions aboutthe flow axis to yield angular calibrated scattering data for particlesthat are non-spherical. The force experienced by a particle in anacoustically driven tube is inherently non-axisymmetric within the crosssection of the flow plane. The acoustic force distribution is dipolar innature for particle focusing to the tube axis yielding force reflectionsymmetries within the plane. Calculations of the acoustic forcepotential U for a particle in a line driven tube in a dipole type modeas one example of this method are shown in FIG. 2( a) where the acousticforce F_(U) can be obtained byF=−∇Uwhere the flow direction is into the page. The in-plane force potentialpossesses reflection symmetry about two planes that intersect thecentral axis. The first plane of symmetry intersects central axis 20 andline drive 22 and the second plane of symmetry lies perpendicular to thefirst. Though two symmetry planes exist within the displayedtwo-dimensional acoustic force potential, only one results in stableequilibrium 24 location with respect to particle rotation. All regularparticles will rapidly rotate into stable equilibrium 24 under smallperturbations within the flow field as shown in FIG. 2( b).

Incorporating a third dimension into the force field calculation (axialcomponent) yields an additional restriction in the rotational freedom ofa particle induced by the acoustic force field. Calculations show thatrod shaped particles (particles with two equal minor axes and one majoraxis) will align their major axis with the axis of the capillary.Particles with bilateral symmetry, e.g. red blood cells, will align onemajor axis parallel to the flow axis and the other major axis parallelto the stable symmetry plane denoted by the white dotted line in FIG. 2(a).

Acoustic Separation of Cells and Cellular Debris

For particle transport to occur in an acoustically driven chamberaccording to one embodiment of the present invention, the acoustic forcemust be large enough to overcome the Brownian motion of the particleswithin the suspending medium. The magnitude of the acoustic radiationpressure force experienced by a particle is directly proportional to theparticle volume, drive level of the acoustic field, mechanicalproperties of the medium and particle, and the spatial gradient of theacoustic field. For this reason, (due to a cubic relationship ofparticle radius) larger particles can be transported in an acousticfield at lower pressure amplitudes and excitation frequencies (smallergradients) than smaller particles. (This is also true for particles thathave a greater relative difference in their mechanical propertiesrelative to the background medium.)

One aspect of one embodiment of an acoustic separation system of thepresent invention is that it can operate clog-free (no filter) withalmost zero pressure drop across the unit. Due to the size dependenceinherent in the acoustic radiation force and thermal particle motion, anembodiment of the present invention can separate samples at the frontend of a flow stream based upon particle size and mechanical contrast.Acoustic forces are used to purify samples by concentrating analytes ofinterest at a specified location for selective collection leavingbackground debris unaffected. Such a system reduces analysis time forsamples with high particulate backgrounds on a flow cytometer by greatlyreducing the particle count and increasing data quality. For instance,Bossuyt showed that cellular debris within whole blood samples preparedby selected lysis methods can yield scattering events that account forup to 80% of all events in CD45 cell counting on a flow cytometer. Maceynoted that certain whole blood lysis methods for preparing lymphocytesfor flow cytometry analysis can result in poor forward and side scatterresolution due to the presence of residual cell fragments. In oneembodiment of the present invention, an in-line purification device,such as a line-driven capillary located just prior to the sample inletof a flow cytometer as shown in FIG. 3, is used to transport largeparticles of interest 30 (e.g. lymphocytes) to central axis 32 of thesample stream while smaller particles 34 such as cellular debris andproteins contained within the lysate remain unaffected. This isespecially true for cellular debris with less mechanical contrast thanthe particles of interest. The central core of the sample stream is thenfed into the flow cytometer and the remaining lysate is discardedeliminating a large particulate concentration from the sample. It shouldbe noted that this method of sample preparation can be used as a samplepurification step for any type of particle/cellular analysis where thereduction of background particulate count is advantageous

Particle Orientation in a Standing Acoustic Wave Field

EXAMPLE 1

To demonstrate the effects of the acoustic field to induce deterministicparticle orientation, experiments using particles with aspect ratiosgreater than unity were conducted with a line driven capillary. In oneexample, the capillary was made of glass and had an by inner diameter ofapproximately 500 μm and an outer diameter of approximately 1000 μm. Anacoustic source was attached to the outer surface of the capillary(parallel with the axis of the capillary) and operated at approximately1.78 MHz and approximately 10 V_(pp). A suspension of circularcylindrical carbon fibers in deionized water was transported down thetube with a syringe pump. The particles were then imaged through amicroscope. The fibers had a minor axial dimension of approximately 8 μmwith varying greater major axis dimensions.

FIG. 4A illustrates the sample as it flowed through the capillary (flowis from left to right). Fibers were seen in random orientations as theywere entrained in the fluid and transported through the system when noacoustic field was present. Upon acoustic excitation of the capillary,the fibers were transported and rotated to align coincident and parallelwith the axis of the capillary, see FIG. 4B. The alignment shown herewas due to the acoustic radiation pressure force aligning the major axisof the particles along the axis of the capillary.

Field-Based Particle Size Selection for In-Line Sample Purification andSeparation/Concentration

By varying the drive voltage and/or the frequency of excitation of theacoustic source in an acoustically driven capillary, a binaryfractionation of particles by size can be achieved. This effect is aresult of the reduced acoustic force felt by the smaller particles dueto the cubic dependence of the acoustic force on particle radius. Inapplication, the larger particles contained within the central core ofthe capillary are fed into a smaller, coaxial capillary discarding theconcentric flow field containing small particulates. The purified samplecan be taken for further sample preparation steps or fed real-time intoa flow cytometer or other means of analysis. Depending upon theapplication, the fluid outside the central core may also be considered avaluable sample to be collected and used for analysis.

EXAMPLE 2

Results from preliminary experiments demonstrating the size selectioncapability as a function of drive level are illustrated in FIGS. 5A-5C.In this example, an acoustically driven capillary was oscillated atapproximately 1.78 MHz. A suspension of latex microspheres containingapproximately 1 μm diameter fluorescent spheres and approximately 10 μmdiameter non-fluorescent spheres are pumped through the drivencapillary. The volume fraction of particles was approximately 2.5×10⁻⁵The capillary is defined by an inner diameter of approximately 500 μmand outer diameter of approximately 1000 μm.

FIG. 5A is a photograph taken through a fluorescence microscope wherethe approximately 10 μm particles are viewed as large circularinclusions and the approximately 1 μm particles are viewed as a grainybackground. (The fluorescence signal from the approximately 1 μmparticles is too low to be observed under the operating conditions ofthe experiment.) Under low acoustic drive level of approximately 7V_(pp) (FIG. 5B), the approximately 10 μm particles rapidly transportedto the axis of the capillary. The approximately 1 μm particles remainedrandomly distributed. Doubling the drive voltage to approximately 16V_(pp) resulted in efficient transport of both sized particles to thecentral axis of the capillary, see FIG. 5C. The bright line along theaxis of the cylinder was a result of the large, local increase influorescence due to the concentration of the approximately 1 μmfluorescent particles at that location.

Acoustic Focusing/Orientation such as the Effects of Reflection Symmetryon Optical Scatter Parameters in an Acoustically Focused Flow Chamber

An embodiment of the present invention addresses angular scatteringassociated with particles that are aligned in the optical scatteringplane as a result of acoustic radiation pressure. The replacement ofhydrodynamic sheath flow with acoustically driven particle alignment ina flow cell preferably leads to improved light scatter data and yieldsnew parameters that are dependent upon particle geometry andorientation. In addition to the importance of particle orientation ofasymmetric biological particles (e.g. RBC's, sperm cells, bacteria) inflow cytometry analysis, complex geometries that are formed from thecoalescence of multiple microspheres or cells (e.g. ‘dumb bell’ shapesfrom agglutination of two particles) also benefit from particleorientation. Particle clusters are preferably more easily discriminatedby having their orientation fixed in the scattering plane. Orientingmicrosphere ‘doublets’ to yield repeatable and unique scatteringsignatures due to how they transit the scattering plane will provide ameans to isolate their contribution in optical scattering data foreither data rejection or data acceptance by utilizing the solution ofthe inverse scattering problem for contacting spheres. The applicationof acoustically oriented particles in a flow stream is also applicableto the field of imaging where viewing selected orientations of particlesis valuable in determining cellular morphology, localization of cellularconstituents, or other particle/cellular characteristics.

Enhanced Detection Capabilities Under Slow-Flow, Stop-Flow, andReverse-Flow Conditions in Acoustically Focused Flow Chambers

Another embodiment of the present invention further addresses theeffects of slow-flow, stop-flow, reverse-flow, and increased analysistimes in flow cytometry,detection that result from replacing sheath flowwith acoustic particle alignment. In the first instance, the ability tostop and reverse the flow direction of the sample stream allows forparticles to be reanalyzed. The flow is stopped and/or reversed usingvarious stop flow and reverse flow devices, including but not limitedto, a pump or one or more valves. Peak spread (increased CV's) and datapoints that are outliers in the analysis plane are system dependentquantities that are a function of laser stability, quality of particlealignment, electronic noise, detector noise, robustness of the assay(on/off rates, etc.), etc. By analyzing a particle of significance morethan once, the data quality can be improved (especially in the case oftransient artifacts) and the statistical uncertainties in rare eventanalysis can be minimized.

Acoustic Field-Based Particle Size Selection for In-Line SamplePurification and Particle Isolation

While acoustic focusing is useful for particle or cell analysis byreplacing sheath flow in the detection system, yet another embodiment ofthe present invention extends the application of acoustic forces inacoustically driven capillaries to particle and/or cell separations forupstream, in-line sample conditioning in flow cytometry systems orgeneral sample preparation and purification. FIG. 6 illustratesutilizing the present invention to acoustically size fractionate (andconcentrate) samples by particle size and/or mechanical contrastrelative to the background medium in real-time at the inlet of a flowcytometer before the analysis stage. Direct fractionation based uponparticle size/mechanical properties alleviates the need for laborintensive sample preparation steps that include centrifugation andfiltering. For flow cytometry applications, this is useful in reducingthe background associated with cellular debris, proteins, and othermolecular components in whole blood assays, and in particular, no-washassays that include cellular lysis. A sample preparation including acellular debris rejection step prior to sample delivery into the flowcytometer can greatly reduce artifacts associated withscatter/fluorescence from the debris.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents. The entire disclosures of allreferences, applications, patents, and publications cited above and/orin the attachments, and of the corresponding application(s), are herebyincorporated by reference.

1. A method for separating particles by size comprising: flowingparticles in a flow stream; applying radial acoustic radiation pressureto the flow stream; and acoustically separating the particles by size byapplying radial acoustic pressure, wherein smaller particles aresubstantially unaffected by the acoustic radiation pressure.
 2. Themethod of claim 1 further comprising transporting larger particles ofthe separated particles into a flow cytometer.
 3. The method of claim 2further comprising analyzing the larger particles.
 4. The method ofclaim 1 wherein acoustically separating the particles by size comprisesacoustically transporting larger particles to a substantially centralaxis of the flow stream.
 5. The method of claim 4 wherein transportingthe larger particles of the separated particles into a flow cytometercomprises flowing the larger particles into an inlet centered around thesubstantially central axis of the flow stream.
 6. The method of claim 1further comprising in-line separating.
 7. The method of claim 1 whereinlarger particles are lymphocytes.
 8. The method of claim 1 whereinacoustically separating the particles is performed without gravity basedseparation.
 9. A method for separating particles by size comprising:flowing particles in a flow stream; applying radial acoustic radiationpressure to the flow stream; acoustically separating the particles bysize by applying radial acoustic pressure, and transporting largerparticles into a flow cytometer, wherein smaller particles comprisecellular debris.
 10. The method of claim 9 further comprising analyzingthe larger particles.
 11. The method of claim 9 wherein acousticallyseparating the particles by size comprises acoustically transporting thelarger particles to a substantially central axis of the flow stream. 12.The method of claim 11 wherein transporting the larger particles into aflow cytometer comprises flowing the larger particles into an inletcentered around the substantially central axis of the flow stream. 13.The method of claim 9 further comprising in-line separating.
 14. Themethod of claim 9 wherein the larger particles are lymphocytes.
 15. Themethod of claim 9 wherein acoustically separating the particles isperformed without gravity based separation.